Binding to the yeast SwI4,6-dependent cell cycle box, CACGAAA, is cell cycle regulated in vivo.

In Saccharomyces cerevisiae commitment to cell division occurs late in the G1 phase of the cell cycle at a point called Start and requires the activity of the Cdc28 protein kinase and its associated G1 cyclins. The Swi4,6-dependent cell cycle box binding factor, SBF, is important for maximal expression of the G1 cyclin and HO endonuclease genes at Start. The cell cycle regulation of these genes is modulated through an upstream regulatory element termed the SCB (SwI4,6-dependent cell cycle box, CACGAAA), which is dependent on both SWI4 and SWI6. Although binding of SWI4 and SWI6 to SCB sequences has been well characterized in vitro, the binding of SBF in vivo has not been examined. We used in vivo dimethyl sulfate footprinting to examine the occupancy of SCB sequences throughout the cell cycle. We found that binding to SCB sequences occurred in the G1 phase of the cell cycle and was greatly reduced in G2. In the absence of either SWI4 or SWI6, SCB sequences were not occupied at any cell cycle stage. These results suggest that the G1-specific expression of SCB-dependent genes is regulated at the level of DNA binding in vivo.     

Five SWI genes are required for expression of the HO gene in yeast

High-frequency mating type interconversion in yeast requires the HO gene, which encodes a site-specific endonuclease that initiates the switching process. We have isolated and analyzed switching-defective mutants. These mutants define five complementation and linkage groups, SWI 1 to SWI 5. We have shown by two assays, Northern hybridization and beta-galactosidase activity in strains containing an HO-lacZ fusion, that mutants defective any SWI gene fail to express the HO gene. In addition, all of the swi mutants exhibit other phenotypes, the most notable being the inviability of double mutants defective in SWI 4 and in either SWI 1, SWI 2 or SWI 3. These results indicate that the SWI genes function in some way as positive regulators of HO expression and have additional cellular roles.     

Oxidant-induced cell-cycle delay in Saccharomyces cerevisiae: the involvement of the SWI6 transcription factor

Cells treated with low doses of linoleic acid hydroperoxide (LoaOOH) exhibit a cell-cycle delay that may provide a mechanism to overcome oxidative stress. Strains sensitive to LoaOOH from the genome-wide deletion collection were screened to identify deletants in which the cell-cycle delay phenotype was reduced. Forty-seven deletants were identified that were unable to mount the normal delay response, implicating the product of the deleted gene in the oxidant-mediated cell-cycle delay of the wild-type. Of these genes, SWI6 was of particular interest due to its role in cell-cycle progression through Start. The swi6 deletant strain was delayed on entry into the cell cycle in the absence of an oxidant, and oxidant addition caused no further delay. Transforming the swi6 deletant with SWI6 on a plasmid restored the G1 arrest in response to LoaOOH, indicating that Swi6p is involved in oxidant sensing leading to cell division delay. Micro-array studies identified genes whose expression in response to LoaOOH depended on SWI6. The screening identified 77 genes that were upregulated in the wild-type strain and concurrently downregulated in the swi6 deletant treated with LoaOOH. These data show that functions such as heat shock response, and glucose transport are involved in the response.